We have demonstrated with electron paramagnetic resonance at organic hydroperoxides are decomposed to free radicals by both human polymorphonuclear leukocytes (PMNS) and purified myeloperoxidase. Peroxyl, alkoxyl, alkyl and the hydroxyl radicals were trapped by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) when organic hydroperoxide was incubated with either PMNs or purified myeloperoxidase. Myeloperoxidase-deficient PMNs produced DMPO radical addict intensities at only about 20-30% of that of normal PMNs. Our studies suggest that myeoperoxidase in PMNs is primarily responsible for the decomposition of organic hydroperoxides to free radicals. The finding of the free radical formation derived from organic hydroperoxides by PMNs may be related to the cytotoxicity of this class of compounds. Soybean lipoxygenase has been shown to catalyze the breakdown of polyunsaturated fatty acid hydroperoxides to produce superoxide radical anion as detected by spin trapping with DMPO. The DMPO adducts of peroxyl, acyl, carbon-centered and hydroxyl radicals were as well identified in incubations containing linoleic acid and lipoxygenase. These DMPO radical adducts were observed just prior to the system becoming anaerobic. The superoxide radical production required the presence of fatty acid substrates, active lipoxygenase and molecular oxygen. Superoxide radical production was inhibited when either nordihydroguaiaretic acid, butylated hydroxytoluene or butylated hydroxyanisole was added to the incubation mixtures. We propose that polyunsaturated fatty acid hydroperoxides are reduced to form alkoxyl radicals and that after an intramolecular rearrangement, the resulting hydroxyalkyl radical reacts with oxygen, forming a peroxyl radical which subsequently eliminates superoxide radical anion.